Compositions and methods for repairing cartilage defects

ABSTRACT

The present disclosure provides compositions and methods for repairing cartilage defects.

RELATED APPLICATIONS

This application is a national stage filing under U.S.C. § 371 of PCTInternational Application No. PCT/US2018/064629 filed Dec. 7, 2018,which claims the benefit of and priority to U.S. Provisional ApplicationNo. 62/596,009, filed Dec. 7, 2017, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND

Chondral and osteochondral lesions such as, for example, focal lesionsin the load bearing region of a knee's articular cartilage, greatlyincrease the risk for osteoarthritis. This type of lesion occursfrequently from, for example, trauma, participation in sports,dissecting osteochondritis, etc. The capacity for spontaneous repair ofchondral lesions is minimal, due in part to the limited blood supply tocartilage tissue.

Certain current therapeutic intervention strategies typically involveremoving damaged or dislodged cartilage from the joint by physicalremoval and washing via arthroscopy. Such treatments typically providetemporarily relief from symptoms of the injury, but they do not treatthe origin of the lesion, or defect, and, in particular do not preventprogressive degradation of the cartilage.

Alternative current therapeutic intervention strategies include totalknee replacements, which are sometimes used to treat severe cases ofosteoarthritis. However, artificial prostheses have a limited lifespanand thus are not optimal for patients at all ages. Moreover, truetreatment of osteoarthritic cartilage degradation requires replacementof defective cartilage with healthy cartilage; autologous chondrocyteimplantation strategies have been described to accomplish suchreplacement. (Brittberg et al. Clin. Orthopaed. Red. Res. (1999) 367S:S147-S155). In such procedures, chondrocytes are harvested from apatient, expanded in cell culture to increase the number ofchondrocytes, and then implanted back into the injury site of thepatient.

More recent work has improved autologous implantation by seedingexpanded autologous cells on a matrix in a process known asmatrix-induced autologous chondrocyte implantation (MACI). (Basad et al.In: Hendrich et al., Cartilage Surgery and Future Perspectives, ThiemeVerlag, 49-56 (2003)).

SUMMARY

The present disclosure provides important improvements to matrix-inducedchondrocyte implantation technologies. For example, among other things,the present disclosure demonstrates feasibility and/or effectiveness ofmatrix-induced implantation of allogeneic, rather than autologous,chondrocytes. Advantages of provided technologies include, for example,that only a single invasive procedure is required to treat subjectsusing provided allogeneic chondrocyte compositions. Even MACI implants,which permit treatment of cartilage lesions and defects using minimallyinvasive interventions, still require at least two interventions, one toharvest autologous cells, and one to administer the implant. Providedtechnologies, thus, represent and embody further improvements even withrespect to MACI technologies.

In some embodiments, the present disclosure provides a compositioncomprising cultured allogeneic cells grown from a cryogenically frozencell bank sample and a resorbable collagen membrane, wherein cells areseeded on the membrane at a density of at least 250,000 cells per cm².

In some embodiments, the present disclosure provides a method ofmanufacturing an allogeneic cartilage matrix, the method comprisingsteps of thawing a cryogenically frozen cell bank sample, culturingcells of the sample, characterizing the sample, preparing a resorbablecollagen membrane, seeding the membrane with culture, and packaging themembrane.

In some embodiments, the present disclosure provides a composition foruse in therapy, wherein the composition comprises cultured allogeneiccells grown from a cryogenically frozen cell bank sample, and aresorbable collagen membrane; wherein the cells are seeded on themembrane at a density of at least 250,000 cells per cm².

In some embodiments, the present disclosure provides a composition foruse in a method of treating a chondral and/or osteochondral defect,wherein the composition comprises cultured allogeneic cells grown from acryogenically frozen cell bank sample and a resorbable collagenmembrane; wherein the cells are seeded on the membrane at a density ofat least 250,000 cells per cm².

In some embodiments, the present disclosure provides a cell bank for usein a method of manufacturing a matrix, wherein the matrix comprisescultured allogeneic cells grown from a cryogenically frozen cell banksample and a resorbable collage membrane; wherein the cells are seededon the membrane at a density of at least 250,000 cells per cm².

In some embodiments, the present disclosure provides a composition foruse in a method of treating a chondral defect and/or osteochondraldefect, wherein the method comprises implanting a composition comprisingcultured allogeneic cells.

In some embodiments, a cryogenic cell bank sample is thawed in a waterbath, a heat block, or a dry cell bath at about 37° C.

In some embodiments, a sample is cultured in medium comprising DMEM. Insome embodiments, a sample is cultured in medium comprising DMEM, HEPES,Fetal Bovine Serum, and Gentamicin.

In some embodiments, a sample is characterized for viability, presenceof viruses, sterility, endotoxin, mycoplasma, senescence, identity,aggrecan, and karyology.

In some embodiments, cells are a monolayer on a membrane.

In some embodiments, cells are dedifferentiated.

In some embodiments, a resorbable collagen membrane comprises type I andtype III collagen. In some embodiments, a resorbable collagen membranecomprises porcine collagen. In some embodiments, porcine collagen isderived from a porcine peritoneum.

In some embodiments, the present disclosure provides a method oftreating a chondral and/or osteochondral defect in a human subject, themethod comprising implanting into the subject a composition provided bythe present disclosure.

In some embodiments, a composition comprises dedifferentiated cells.

In some embodiments, a human subject is an adult or juvenile.

In some embodiments, a chondral defect and/or osteochondral defect is inan articulating joint. In some embodiments, an articulating joint is aknee, hip, ankle, elbow, wrist or shoulder.

In some embodiments, a composition is implanted over and/or into adefect.

In some embodiments, a composition is trimmed to a size and/or shape tocover over and/or fit into a chondral and/or osteochondral defect.

In some embodiments, a composition is implanted with at least onesurface comprising allogeneic human chondrocytes in contact with achondral and/or osteochondral defect.

In some embodiments, multiple layers of a composition are implanted overand/or into a defect.

In some embodiments, cells comprise chondrocytes.

In some embodiments, the present disclosure provides cell bankscomprising cultured allogeneic human chondrocytes at a density of atleast 250,000 cells per cm², wherein cultured chondrocytes arecryogenically frozen and characterized by a quality control assay.

In some embodiments, a cell bank comprises human cadaver chondrocytes.In some embodiments, a cell bank comprises primary cultures. In someembodiments, a cell bank comprises secondary cultures.

In some embodiments, a quality control assay comprises one or more ofagarose, six well plate, and cell pellet assays.

In some embodiments, the present disclosure provides methods ofpreparing a cell bank, wherein the methods comprise steps of obtaining atissue sample from a human, inspecting a tissue sample forcontamination, weighing a tissue sample, mincing a tissue sample,digesting a tissue sample, counting cells in a tissue sample todetermine viability, culturing cells; and/or, cryogenically freezingcells for storage.

In some embodiments, methods comprise a step of obtaining a tissuesample comprising removing cartilage from a human cadaver. In someembodiments, methods comprise a step of obtaining a tissue samplecomprises removing cartilage from a human cadaver within about 24 hoursto about 7 days after death. In some embodiments, methods comprise astep of obtaining a tissue sample comprises removing cartilage from ahuman cadaver within about 24 hours after death.

In some embodiments, a tissue sample is inspected for synovium, bone,fibrous tissue, fatty tissue, and/or contamination.

In some embodiments, a tissue sample weighs between about 1 g and about9 g. In some embodiments, a tissue sample weighs about 9 g. In someembodiments, a tissue sample is divided into pieces weighing betweenabout 200 mg and about 400 mg. In some embodiments, a tissue sample isdivided into pieces weighing between about 200 mg and about 300 mg. Insome embodiments, a tissue sample is divided into pieces weighingbetween about 280 mg and about 300 mg. In some embodiments, a tissuesample is divided into pieces weighing about 300 mg.

In some embodiments, tissue is digested with a protease. In someembodiments, tissue is digested with collagenase.

In some embodiments, a tissue sample is minced into pieces from about0.5 mm² to about 3 mm². In some embodiments, a tissue sample is mincedinto pieces from about 0.5 mm² to about 2 mm². In some embodiments, atissue sample is minced into pieces from about 0.5 mm² to about 1 mm².In some embodiments, a tissue sample is minced into pieces of about 0.5mm².

In some embodiments, cells are counted with a hemacytometer. In someembodiments, a tissue sample comprises from about 50% to about 100%viable cells. In some embodiments, a tissue sample comprises from about70% to about 100% viable cells. In some embodiments, a tissue samplecomprises about 70% viable cells.

In some embodiments, cells are cultured in medium comprising DMEM. Insome embodiments, a sample is cultured in medium comprising DMEM, HEPES,Fetal Bovine Serum, and Gentamicin. In some embodiments, cells arecultured at about 37° C.

In some embodiments, cells are cryogenically frozen at about −80° C. forabout 2 to 24 hours. In some embodiments, cells are cryogenically frozenand stored in liquid nitrogen.

In some embodiments, the present disclosure provides methods ofcharacterizing a cell bank sample. In some embodiments, the methodscomprise a step of thawing cryogenically frozen primary cell banksamples, culturing secondary samples from primary samples, culturingtertiary samples from secondary samples; and/or assaying samples todetermine cell viability, mycoplasma, endotoxin, sterility, senescence,identity, and aggrecan values.

In some embodiments, samples are assayed using an agarose assay. In someembodiments, an agarose assay tests for percentage of cells formingcolonies for greater than or equal to two divisions. In someembodiments, an acceptance criterion of the agarose assay comprises atleast 6.8% of cells form colonies for at least two divisions.

In some embodiments, samples are assayed using a six well plate assay.In some embodiments, a six well plate assay tests for an average numberof cells per well. In some embodiments, an acceptance criterion of thesix well assay comprises an average of cells per well that is greaterthan or equal to 0.88×10⁵.

In some embodiments, samples are assayed using a pellet culture assay.In some embodiments, a pellet culture assay tests for ability of cellsto generate cartilage following cellular culture expansion. In someembodiments, an acceptance criterion of the pellet culture assaycomprises dga greater than or equal to −4.73 Ct.

In some embodiments, an acceptance criterion for the assay formycoplasma comprises no detectable level.

In some embodiments, an acceptance criterion for the assay for sterilitycomprises no detection of growth.

In some embodiments, an acceptance criterion for cell viabilitycomprises at least 70% viable cells.

In some embodiments, an acceptance criterion for senescence comprisescultures do not senesce in less than 5 passages or cultures are notimmortal.

In some embodiments, an acceptance criterion for identity comprisesidentification of the presence of chondrocytes.

In some embodiments, the present disclosure provides methods ofculturing a cell bank sample. In some embodiments, the methods comprisea step of thawing a cryogenically frozen cell bank sample, addingculture medium to a flask, counting cells to determine viability,growing cells in a cell culture medium, feeding a cell culture, and/ortreating a cell culture with trypsin.

In some embodiments, a cryogenically frozen cell bank sample is thawedin a water bath, a heat block, or a dry cell bath at about 37° C.

In some embodiments, a sample is cultured in medium comprising DMEM.

In some embodiments, cells are counted with a hemacytometer.

In some embodiments, a cell culture is fed at least every 1 to 4 days.

In some embodiments, a cell culture is treated with 0.05% trypsinsolution. In some embodiments, a cell culture is treated with trypsinsolution until cells are detached from the flask.

BRIEF DESCRIPTION OF THE DRAWING

The present teachings described herein will be more fully understoodfrom the following description of various illustrative embodiments, whenread together with the accompanying drawing. It should be understoodthat the drawing described below is for illustration purposes only andis not intended to limit the scope of the present teachings in any way.

FIG. 1 depicts an exemplary series of photographs showing cartilageregeneration using both autologous and allogeneic chondrocytes.

FIG. 2 depicts an exemplary series of histological samples showingcartilage regeneration using both autologous and allogeneicchondrocytes.

DEFINITIONS

As used herein, the term “about,” as used in reference to a value,refers to a value that is similar, in context to the referenced value.In general, those skilled in the art, familiar with the context, willappreciate the relevant degree of variance encompassed by “about” inthat context. For example, in some embodiments, the term “about” mayencompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%,15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, orless of the referred value.

As used herein, the term “adult” refers to a human seventeen years ofage or older. In some embodiments, a human adult has a weight within therange of about 90 pounds to about 250 pounds.

As used herein, the term, “associated with” refers to two events orentities when presence, level and/or form of one is correlated with thatof the other. For example, a particular entity (e.g., polypeptide,genetic signature, metabolite, microbe, etc.) is considered to beassociated with a particular disease, disorder, or condition, if itspresence, level and/or form correlates with incidence of and/orsusceptibility to a disease, disorder, or condition (e.g., across arelevant population). In some embodiments, two or more entities arephysically “associated” with one another if they interact, directly orindirectly, so that they are and/or remain in physical proximity withone another. In some embodiments, two or more entities that arephysically associated with one another are covalently linked to oneanother; in some embodiments, two or more entities that are physicallyassociated with one another are not covalently linked to one another butare non-covalently associated, for example by means of hydrogen bonds,van der Waals interaction, hydrophobic interactions, magnetism, andcombinations thereof.

As used herein, the term “biocompatible” refers to materials that do notcause significant harm to living tissue when placed in contact with suchtissue, e.g., in vivo. In certain embodiments, materials are“biocompatible” if they are not toxic to cells. In certain embodiments,materials are “biocompatible” if their addition to cells in vitroresults in less than or equal to 20% cell death, and/or theiradministration in vivo does not induce significant inflammation or othersuch adverse effects.

As used herein, the term “chondrocytes” or “cartilage cells,” refers tocells that are capable of expressing biochemical markers characteristicof chondrocytes, including but not limited to type II collagen,aggrecan, chondroitin sulfate and/or keratin sulfate. In someembodiments, chondrocytes, or cartilage cells, express morphologicmarkers characteristic of smooth muscle cells, including but not limitedto a rounded morphology in vitro. In some embodiments, chondrocytes, orcartilage cells, are able to secrete type II collagen in vitro. In someembodiments, chondrocytes, or cartilage cells, are able to secreteaggrecan in vitro. In some embodiments, chondrocytes, or cartilagecalls, are able to generate tissue or matrices with hemodynamicproperties of cartilage in vitro.

As used herein, the term “density” refers to an average number of asubstance, for example, cells or another object, per unit area ofvolume. In some embodiments, density is cell density, i.e., number ofcells per unit of surface area. In some embodiments, an average densityis approximated by dividing a number of cells seeded by a macroscopicsurface are of a surface on which they are grown. In some embodiments, asurface is two-dimensional. In some embodiments, a surface isthree-dimensional.

As used herein, the term “inoculating” refers to a process or stepwhereby cells are brought into contact with a surface, for example asurface of a container suitable for cell culture. In some embodiments,cells inoculated onto a cell culture surface (e.g., flask, dish) adherefor a period of time. In some embodiments, once inoculated onto a cellculture surface, cells proliferate. In some embodiments, cells (e.g.,chondrocytes) inoculated onto a cell culture surface mayde-differentiate. In some embodiments, cells (e.g., chondrocyteprecursors, mesenchymal stem cells) inoculated onto a cell culturesurface may differentiate into a desired cell type, e.g., chondrocytes.

As used herein the term “in vitro” refers to events that occur in anartificial environment, e.g., in a test tube or reaction vessel, in cellculture, etc., rather than within a multi-cellular organism.

As used herein the term “in vivo” refers to events that occur within amulti-cellular organism, such as a human and a non-human animal. In thecontext of cell-based systems, the term may be used to refer to eventsthat occur within a living cell (as opposed to, for example, in vitrosystems).

As used herein, the term “medium” refers to components which supportgrowth or maintenance of cells in culture. In some embodiments, this mayinclude traditional liquid cell culture medium and an additional factor.In some embodiments, additional factors may include, for example, serum,antibiotics, growth factors, pharmacological agents, buffers, pHindicators and the like. In some embodiments, medium may be used in aprocess to isolate cells (e.g., chondrocytes and/or chondrocyteprecursors) from a tissue sample (e.g., a cartilage sample). In someembodiments, tissue is mechanically disrupted (e.g., chopped, minced,blended) then combined with medium. In some embodiments, mediumcomprises enzymes (e.g., collagenase, protease) to digest tissue andrelease cells.

As used herein, the term “conditioned medium” refers to medium which hasbeen contacted with cells to allow for the composition of medium to bemodified, for example by uptake or release of one or more metabolites,nutrients, or factors.

As used herein, the term “patient” refers to any organism to which aprovided composition is or may be administered, e.g., for experimental,diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typicalpatients include animals (e.g., mammals such as mice, rats, rabbits,non-human primates, and/or humans). In some embodiments, a patient is ahuman. In some embodiments, a patient is suffering from or susceptibleto one or more disorders or conditions. In some embodiments, a patientdisplays one or more symptoms of a disorder or condition. In someembodiments, a patient has been diagnosed with one or more disorders orconditions. In some embodiments, the patient is receiving or hasreceived certain therapy to diagnose and/or to treat a disease,disorder, or condition.

As used herein, the term “seeding” refers to a process or step wherebycells are brought into contact with a support matrix, and adhere (withor without an adhesive) to a support matrix (e.g., a collagen membrane)for a period of time. Seeded cells may divide and/or differentiate on asupport matrix. In some embodiments, cells are seeded onto a supportmatrix prior to being implanted into a subject.

As used herein, the term “subject” refers to an organism, typically amammal (e.g., a human, in some embodiments including prenatal humanforms). In some embodiments, a subject is suffering from a relevantdisease, disorder or condition. In some embodiments, a subject issusceptible to a disease, disorder, or condition. In some embodiments, asubject displays one or more symptoms or characteristics of a disease,disorder or condition. In some embodiments, a subject does not displayany symptom or characteristic of a disease, disorder, or condition. Insome embodiments, a subject is someone with one or more featurescharacteristic of susceptibility to or risk of a disease, disorder, orcondition. In some embodiments, a subject is a patient. In someembodiments, a subject is an individual to whom diagnosis and/or therapyis and/or has been administered. In some embodiments, a subject is adonor of a biological sample, tissue and/or material.

As used herein, the term “substantially” refers to the qualitativecondition of exhibiting total or near-total extent or degree of acharacteristic or property of interest. One of ordinary skill in thebiological arts will understand that biological and chemical phenomenararely, if ever, go to completion and/or proceed to completeness orachieve or avoid an absolute result. The term “substantially” istherefore used herein to capture the potential lack of completenessinherent in many biological and chemical phenomena.

As used herein, the term “substantially free of endotoxin” refers to alevel of endotoxin per dose of a composition that is less than isallowed by the FDA for a biologic product (i.e., total endotoxin of 5EU/kg body weight per hour, which for an average 70 kg person is 350 EUper total dose).

As used herein, the term “substantially free of mycoplasma and/ormicrobial contamination” refers to a negative reading for a generallyaccepted test of contamination known to those skilled in the art. Forexample, mycoplasma contamination is determined by subculturing aproduct sample in broth medium and distributing the culture over agarplates on days 1, 3, 7, and 14 at 37° C. with appropriate positive andnegative controls. In some embodiments, mycoplasma contamination isdetermined using a real-time PCR method. The product sample appearanceis compared microscopically at 100×, to that of a positive and negativecontrol. Additionally, presence of mycoplasma contamination may bedetected by inoculation of an indicator cell culture, which is incubatedfor 3 and 5 days then examined at 600× by epifluorescence microscopyusing a DNA-binding fluorochrome. The composition is consideredsatisfactory if agar and/or broth media procedure and indicator cellculture procedure show no evidence of mycoplasma contamination. In someembodiments, an assay that may be utilized to assess a level ofmicrobial contamination may be or comprise the U.S. Pharmacopeia (USP)Direct Transfer Method. This involves inoculating a sample into a tubecontaining tryptic soy broth media and fluid thioglycollate media. Tubesare observed periodically for a cloudy appearance (turbidity) during aspecified period (e.g., 14 days) of incubation. A cloudy appearance onany day in either medium indicates contamination, with a clearappearance (no growth) indicating that a composition may be consideredto be substantially free of contamination. In some embodiments, anapproved alternative to a USP method for detection of microbialcontamination is used, for example, a BacT/ALERT test using differentmedia formulations.

As used herein, the term “surface area” refers to, for example, squarearea, cm², or to the macroscopic surface area of a substrate.

As used herein, the term “treatment” (also “treat” or “treating”) refersto administration of a therapy that partially or completely alleviates,ameliorates, relives, inhibits, delays onset of, reduces severity of,and/or reduces incidence of one or more symptoms, features, and/orcauses of a particular disease, disorder, and/or condition. In someembodiments, such treatment may be of a subject who does not exhibitsigns of the relevant disease, disorder and/or condition and/or of asubject who exhibits only early signs of the disease, disorder, and/orcondition. Alternatively or additionally, such treatment may be of asubject who exhibits one or more established signs of the relevantdisease, disorder and/or condition. In some embodiments, treatment maybe of a subject who has been diagnosed as suffering from the relevantdisease, disorder, and/or condition. In some embodiments, treatment maybe of a subject known to have one or more susceptibility factors thatare statistically correlated with increased risk of development of therelevant disease, disorder, and/or condition.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present disclosure provides certain compositions comprisingchondrocytes, particularly of human origin, and various related methods(e.g., methods of use and/or of manufacture) of such compositions and/orassociated technologies. In particular, the present disclosure providescompositions comprising allogeneic human chondrocytes which compositionsmay be useful, for example, for treatment of chondral and/orosteochondral lesions (e.g., for example, focal lesions in the loadbearing region of a knee's articular cartilage). That is, the presentdisclosure provides compositions comprising human chondrocytes from afirst human subject for use in treating damage in a second humansubject, different from the first human subject.

In some embodiments, in accordance with the present disclosure, humanchondrocytes harvested from a first human subject are cultured ex vivo(e.g., in vitro), and are seeded onto a resorbable support matrix (e.g.,a collagen membrane), that may be implanted into a second human subject.

Among other things, the present disclosure provides technologies forproducing cultured preparations (e.g., suspensions) of allogeneic humanchondrocytes, including preparations that display certaincharacteristics (e.g., cell yield, cell suspension density, viability,sterility etc.), and/or technologies for preparing, storing,transporting and/or utilizing such cultured preparations.

In some embodiments, the present disclosure provides technologies forproducing compositions in which cultured allogeneic human chondrocytecells are seeded onto a resorbable support matrix according to certainparameters (e.g., membrane integrity, cell viability, cell identity,sterility), and/or technologies for preparing (e.g., performingseeding), storing, transporting and/or utilizing such compositions.

In some embodiments, the present disclosure provides technologies thatpermit and/or achieve treatment of clinically significant chondraland/or osteochondral lesions, defects, injuries and/or trauma. In someembodiments, treatment comprises tissue repair and/or regeneration.

In some embodiments, compositions comprising chondrocytes are implantedinto a subject at or near a site of a lesion, defect, injury and/ortrauma, for example, at or near an articular surface. Articular surfacesthat may be treated using the compositions of the present disclosureinclude articular surfaces of, for example, a knee, ankle, wrist, hip,elbow or shoulder.

In some embodiments, the present disclosure provides compositions,methods, and uses of a cell bank. In some embodiments, a cell bank iscreated from human chondrocytes harvested from a first human subjectthat are cultured ex vivo (e.g., in vitro), and cryogenically frozen.

Compositions

Compositions of the present disclosure comprise human chondrocytesexpanded in culture and seeded onto a support matrix.

Cell Preparations

The present disclosure utilizes allogeneic human chondrocytes forpreparation of useful compositions as described herein. Typically,allogeneic human chondrocytes are isolated from tissue of a firstsubject, who is a different subject from that into whom providedcompositions will be implanted.

In some embodiments, allogeneic human chondrocytes are obtained fromtissue harvested from a human. In some embodiments, allogeneic humanchondrocytes are obtained from tissue harvested from an adult human.Alternatively or additionally, in some embodiments, allogeneic humanchondrocytes are obtained from tissue harvested from a cadaver. In someembodiments, allogeneic human chondrocytes are obtained from a cellbank.

Harvested tissue is typically subjected to one or more processing stepsso that a source cell preparation comprising chondrocytes andchondrocyte precursors may be isolated.

Such a source cell preparation is utilized to prepare a culturedallogeneic human chondrocyte preparation for use in accordance with thepresent disclosure. Those skilled in the art are aware that humanchondrocyte cells typically express certain detectable markers such as,for example, HAPLN1. See, for example, U.S. Pat. No. 8,029,992, whichdescribes certain markers expressed on cultured human chondrocyte cellsand whose teachings in that regard are incorporated herein by reference.

In some embodiments, a preparation of human chondrocytes useful inaccordance with the present disclosure is characterized by expressionlevels of one or more relevant markers by cells within a preparation.For example, in some embodiments, one or more chondrocyte markers arepresent at a level above a particular threshold in a preparation.Alternatively or additionally, in some embodiments, one or more markersof a non-chondrocyte cell type (e.g., one or more fibroblast and/orsynoviocyte markers) are present at a level below a particular thresholdin a preparation (e.g., MFAP5). Those skilled in the art will befamiliar with techniques for determining marker level (e.g., detectionof RNA and/or protein according to known technologies).

In some embodiments, RNA expression levels for genes overexpressed bychondrocytes (e.g., HAPLN1) are measured in cultured cells. In someembodiments, RNA expression for genes overexpressed by synoviocytes(e.g., MFAP5) is measured in cultured cells. In some embodiments, RNAexpression levels are presented as a ratio of expression of achondrocyte marker (e.g., HAPLN1) versus expression of a synoviocytemarker (MFAP5). In some embodiments, cultured chondrocytes demonstraterelative RNA expression levels (HAPLN1 vs. MFAP5) of about −2, about −1,about 0, about +1, about +2, about +3, about +4, about +5, about +6,about +7, about +8 about +9, about +10 or more on a log scale. In someembodiments, cultured chondrocytes demonstrate relative RNA expressionlevels ranging from about −2 to about +10, about −1 to about +9, about 1to about 10, about +3 to about +8, about +5 to about +7 or rangestherein. In some embodiments, cultured synoviocytes demonstrate relativeRNA expression levels of about less than −2 on a log scale. In someembodiments, cultured synoviocytes demonstrate relative RNA expressionlevels ranging from less than −2 to −10 on a log scale.

In some embodiments, chondrocytes prepared from a source cellpreparation are present in culture at a density sufficient to seed asupport matrix with at least 250,000 cells/cm². In some embodiments,chondrocytes expanded in culture are dedifferentiated when present in amonolayer culture. In some embodiments, dedifferentiated chondrocytesexhibit a fibroblastic phenotype. In some embodiments, dedifferentiatedchondrocytes down regulate expression of a gene encoding ECM, forexample, ACAN and/or COL2A1. In some embodiments, dedifferentiatedchondrocytes produce and/or secrete a lesser amount of ECM, for example,collagen (e.g., type II collagen) and/or aggrecan (also known ascartilage-specific proteoglycan core protein or chondroitin sulfateproteoglycan 1). Without wishing to be bound by theory,de-differentiation occurs after removal of chondrocytes from3-dimensional cartilage matrix and is observed during expansion of cellsin monolayer culture.

In some embodiments, chondrocyte preparations disclosed herein comprisea sufficient number of cells to seed a support matrix. In someembodiments, chondrocyte preparations comprise at least about 3×10⁶,4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶ or more cells following asecond passage. In some embodiments, chondrocyte preparations compriseat least about 3×10⁶ cells after a second passage. In some embodiments,chondrocyte preparations disclosed herein comprise at least about 1×10⁷,2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷ or more cells ata final passage. In some embodiments, chondrocyte preparations disclosedherein comprise at least 1×10⁷ cells at a final passage.

In some embodiments, chondrocyte cultures are about 50%, 60%, 70%, 80%,90%, 95%, 98% or more confluent. In some embodiments, chondrocytecultures are about 100% confluent. In some embodiments, chondrocytecultures are about 50% to 90% confluent.

In some embodiments, chondrocytes are seeded on a support matrix atdensity of at least 250,000 cells/cm², 300,000 cells/cm², 400,000cells/cm², 500,000 cells/cm², 600,000 cells/cm², 700,000 cells/cm²,800,000 cells/cm², 900,000 cells/cm², 1,000,000 cells/cm², or more.

Among other things, the present disclosure provides cell preparations inwhich a significant percentage of cells are viable; such high viabilitycell preparations can materially improve, and may be required for,successful treatment of a particular lesion or defect. In someembodiments, at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or more of cellspresent in a preparation are viable. In some embodiments, at least 90%of chondrocytes in a preparation are viable.

In some embodiments, a composition of the disclosure provided herein issubstantially free of components used during preparation of a sourcecell preparation and during expansion of chondrocytes (e.g., fetalbovine serum albumin, fetal bovine serum and/or horse serum). Forexample, in some embodiments, a composition provided herein comprisesless than 10 μg/ml, 5 μg/ml, 4 μg/ml, 3 μg/ml, 2 μg/ml, 1 μg/ml, 0.05μg/ml fetal bovine serum albumin. In some embodiments, a cellpreparation is substantially free of mycoplasma, endotoxin, and/ormicrobial (e.g., aerobic microbe(s), anaerobic microbes(s) and/or fungi)contamination. In some embodiments, a cell preparation tests negativefor mycoplasma, endotoxin and/or microbial contamination.

Support Matrix

A support matrix for use in accordance with the present disclosure ismade of a material to which human allogeneic chondrocyte cells adhere.In some embodiments, a support matrix comprises and/or is coated with anadhesive agent that facilitates and/or enables cell adherence.

In some embodiments, a support matrix supports cell proliferation.

In some embodiments, a support matrix is bioresorbable. In some suchembodiments, a bioresorbable matrix may degrade over a period of hours,days, weeks or months. For example, a bioresorbable matrix may degradewithin at least 24 hours, at least 7 days, at least 30 days or at least6 months. In some embodiments, a support matrix may act as a hemostaticbarrier inhibiting penetration of adjacent cells and tissues into aparticular area of the body, for example, an area requiring treatment(e.g., an articular joint).

In some embodiments, a support matrix is a gel, a solid, or asemi-solid. In some embodiments, a support matrix is impermeable,permeable or semi-permeable (e.g., comprising pores). In someembodiments, a support matrix is comprised of a synthetic material, anatural material, or a combination thereof.

In some embodiments, a support matrix may have a structure thatcomprises a membrane, microbead, fleece, thread, gel or combinationthereof.

In some embodiments, a support matrix may be or comprise biologicalmaterial generated by cells; in some such embodiments, a biologicalmaterial is generated by cells in culture. Alternatively, in some suchembodiments, a biological material is generated by cells in tissue(e.g., in vivo). In some embodiments, such biological material isgenerated by cells that are allogeneic to a subject who will receivetreatment as described herein.

In some embodiments, a support matrix may be or comprise collagen. Forexample, a support matrix may be or comprise type I collagen, type IIcollagen, type III collagen, or a combination thereof (e.g., may includea combination of type I collagen and type II collagen, or may include acombination of type I collagen and type III collagen). In someembodiments, a support matrix is comprised of primarily type I collagenon a first side and type III collagen on a second side. In someembodiments, a first side of a support matrix comprising type I collagenis a smooth surface. In some embodiments, a second side of a supportmatrix comprising type III collagen is a rough surface. In someembodiments, a rough surface of a support matrix is suitable for cellseeding. In some embodiments, a smooth surface of a support matrix issuitable to contact a joint surface.

In some embodiments, some or all collagen in a support matrix for use inaccordance with the present disclosure may be cross-linked; in someembodiments, it may be uncross-linked.

In some embodiments, collagen utilized in accordance with the presentdisclosure is derived from an animal such as a pig. In some embodiments,collagen is derived from the peritoneum of a pig.

In some embodiments as described herein, a support matrix comprises acombination of type I and type III porcine collagen.

In some embodiments, cells (e.g., chondrocytes) seeded onto and/orcultured on a support matrix as described herein may produce one or moreextracellular matrix proteins (e.g., collagen) that interact with and/orbecome incorporated into, a support matrix

In some embodiments, a support matrix may also include proteins,polypeptides, hyaluronic acid) and/or polymers (e.g., elastin, fibrin,laminin, fibronectin). In some embodiments, a support matrix iscell-free.

In some embodiments, a support matrix has a surface area, size, shape,and/or dimension appropriate for treatment of a particular chondral orosteochondral defect, lesion or injury. In some embodiments, a supportmatrix is provided in a form (e.g., a sheet form) that is readily shaped(e.g., by folding, cutting, trimming etc.) for administration to aparticular chondral or osteochondral defect.

In some embodiments, a surface area of a support matrix is at leastabout 5 cm², 10 cm², 12 cm², 13 cm², 13.5 cm², 14 cm², 14.5 cm², 15 cm²,15.5 cm², 16 cm², 17 cm², 18 cm², 19.5 cm², 20 cm², 20.5 cm², 21.5 cm²,22 cm², 25 cm², 30 cm² or larger. A dimension of a support matrix may beany dimension necessary to achieve a desired surface area suitable fortreating a chondral and/or osteochondral defect. For example, dimensionsof a 20 cm² support matrix may be about 2 cm×10 cm, 2.5 cm×8 cm, 3cm×6.7 cm or 4 cm×5 cm. In some embodiments, a surface area of a supportmatrix (e.g., collagen membrane) may be about 14.5 cm² with dimensionsof about 3 cm×5 cm. In some embodiments, a surface area of a supportmatrix (e.g., collagen membrane) may be about 20 cm² with dimensions ofabout 4×5 cm².

Cell Seeded Support Matrix

Among other things, the present disclosure provides compositionscomprising cultured human chondrocytes seeded onto a support matrix(e.g., collagen membrane).

Typically, cells that have been cultured for a period of time (e.g., 3days to 5 weeks) are present on or in a support matrix. In someembodiments, cells seeded onto a support matrix are adherent. In someembodiments, cells are adherent to a support matrix to an extent thatthey do not wash off a matrix during subsequent cell culturing steps,are not displaced from a matrix during transported and/or not displacedfrom a matrix during a surgical procedure to implant a matrix.

Among other things, the present disclosure provides cell seeded supportmatrices in which a significant percentage of cells are viable; suchhigh viability of cells present on a cell seeded matrix can materiallyimprove, and may be required for, successful treatment of a particularlesion or defect. In some embodiments, at least 70%, 75%, 80%, 85%, 90%,95%, 98% or more of cells present on a cell seeded matrix are viable. Insome embodiments, at least 90% of chondrocytes present on a cell seedmatrix are viable.

Cells seeded onto a cell seeded support matrix are viable for at leastabout 1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks or more. Insome embodiments, cells seeded onto a support matrix divide. In someembodiments, a cell seeded support matrix is stored at about 4° C. toabout 37° C.

In some embodiments, a cell seeded support matrix comprises at least250,000 cells/cm², 300,000 cells/cm², 400,000 cells/cm², 500,000cells/cm², 600,000 cells/cm², 700,000 cells/cm², 800,000 cells/cm²,900,000 cells/cm², 1,000,000 cells/cm², or more. In some embodiments, acell seeded matrix comprising greater than 250,000 cells/cm² 300,000cells/cm², 400,000 cells/cm², 500,000 cells/cm², 600,000 cells/cm²,700,000 cells/cm², 800,000 cells/cm², 900,000 cells/cm², 1,000,000cells/cm² is suitable for implant into a subject.

In some embodiments, a cell seeded support matrix comprises at least5×10⁶, 7.5×10⁶, 1.0×10⁷, 1.5×10⁷, 2.0×10⁷, 2.5×10⁷, 3.0×10⁷ or morecells. In some embodiments, a 20 cm² porcine type I and type IIIcollagen membrane comprises about 1.0×10⁷ chondrocytes to about 2.0×10⁷chondrocytes. In some embodiments, a 14.5 cm² porcine type I and typeIII collagen membrane comprises about 7.5×10⁶ chondrocytes to about1.5×10⁷ chondrocytes.

In some embodiments, a cell seeded support matrix may also comprisemedium (e.g., DMEM) and supplements (e.g., fetal bovine serum,antibiotic). In some embodiments, medium comprises about 7%, about 8%,about 9%, about 10%, about 11% fetal bovine serum. In some embodiments,medium is supplemented with 8.9%+/−0.2% fetal bovine serum andgentamicin.

In some embodiments, a cell seeded support matrix has a surface area ofat least about 5 cm², 10 cm², 12 cm², 13 cm², 13.5 cm², 14 cm², 14.5cm², 15 cm², 15.5 cm², 16 cm², 17 cm², 18 cm², 19.5 cm², 20 cm², 20.5cm², 21.5 cm², 22 cm², 25 cm², 30 cm². In some embodiments, a cellseeded support matrix has a surface area of about 20 cm² (e.g., 4 cm×5cm). In some embodiments, a cell seeded support matrix has a surfacearea of about 14.5 cm² (e.g., about 3 cm×5 cm). In some embodiments, acell seeded support matrix is trimmed, shaped, cut, molded or formed andcorresponds to a shape of a defect, lesion and/or injury in need oftreatment. In some embodiments, a cell seeded support matrix is of anirregular shape.

In some embodiments, a cell seeded support matrix is substantially freeof components used during preparation of a source cell preparation ofduring expansion of chondrocytes (e.g., fetal bovine serum albumin,fetal bovine serum and/or horse serum). For example, in someembodiments, a cell seeded support matrix provided herein comprises lessthan 10 μg/ml, 5 μg/ml, 4 μg/ml, 3 μg/ml, 2 μg/ml, 1 μg/ml, 0.05 μg/mlfetal bovine serum albumin. In some embodiments, a cell seeded supportmatrix is substantially free of mycoplasma, endotoxin, and/or microbial(e.g., aerobic microbe(s), anaerobic microbes(s) and/or fungi)contamination.

In some embodiments, a cell seeded support matrix composition, preparedin accordance with the present disclosure, comprises a biocompatibleadhesive or glue. In some embodiments, a least a portion of a cell iscoated with a biocompatible adhesive or glue. In some embodiments, abiocompatible adhesive or glue forms a layer over cells on a supportmatrix. In some embodiments, a biocompatible adhesive or glue forms alayer under cells on a support matrix. In some embodiments, a cellseeded support matrix comprises multiple layers of biocompatibleadhesive or glue and cells. In some embodiments, a biocompatibleadhesive or glue is impregnated within a support matrix.

In some embodiments, the present disclosure provides for cells and glue,and/or adhesive, combined together in a mixture of one or morealternating layers of cells and glue, and/or adhesive, on a surface oredge of a support matrix.

In some embodiments, biocompatible adhesives or glues used incompositions of the disclosure include an organic fibrin glue (e.g.,TISSEEL®, fibrin based adhesive available from Baxter, Austria) or afibrin glue prepared during surgery using autologous blood.

Methods of Preparation

In some embodiments, allogeneic human chondrocytes are obtained fromtissue harvested from a human. In some embodiments, allogeneic humanchondrocytes are obtained from tissue harvested from an adult human. Insome embodiments, a source tissue sample may be obtained from a humansubject who is living. Alternatively or additionally, in someembodiments, allogeneic human chondrocytes are obtained from tissueharvested from a cadaver. When a sample is obtained from a humancadaver, a sample may be collected within 24 hours of death oralternatively, up to 1 week after death.

In some embodiments, cells of the composition may be obtained from ahuman subject by biopsy, resection, excision and/or dissection of asource tissue sample comprising chondrocytes or chondrocyte precursors.In some embodiments, bone is included in a tissue sample because itincreases likelihood or number of viable cells. In some embodiments, ahuman subject is an adult. In some embodiments, an adult is about 18 to30 years of age, 18 to 40 years of age, 18 to 50 years of age, 18 to 55years of age or 18 to 60 years of age. In some embodiments, a sample isobtained from an adult about 20 to 30 years of age. In some embodiments,a human subject is a juvenile. In some embodiments, a human subject isabout 1 to 17 years of age. In some embodiments a human subject is about12 to 39 years of age. In some embodiments a human subject is at least10 years of age. The present disclosure contemplates that a sourcetissue sample comprising chondrocytes, or chondrocyte precursors, isobtained from a human subject other than a subject(s) in need oftreatment (e.g., cells are allogeneic).

In some embodiments, a tissue sample is subject to inspection to ensurephysical integrity. In some embodiments, the inspection is visual. Insome embodiments, a tissue sample is inspected to determine the mediatype it is transported in. In some embodiments, a tissue sample isinspected to determine if the tissue was received in frozen media. Insome embodiments, a tissue sample is inspected to determine if foreignmatter is present. In some embodiments, a tissue sample is inspected todetermine if the transport media is expired. In some embodiments,damaged cartilage is segregated. In some embodiments, abnormal cartilageis segregated. In some embodiments, damaged cartilage is rejected. Insome embodiments, abnormal cartilage is rejected.

In some embodiments, a tissue sample is inspected for synovium. In someembodiments, a tissue sample is inspected for bone. In some embodiments,a tissue sample is inspected for unwanted tissue. In some embodiments, atissue sample is inspected for areas with a lack of rigidity. In someembodiments, a tissue sample is inspected by prodding each side todetect one or more areas that lack rigidity. In some embodiments atissue sample is inspected for membranous tissue. In some embodiments, atissue sample is inspected for fibrous tissue. In some embodiments, atissue sample is inspected for fatty tissue. In some embodiments,presence of one or more of bone, synovium, thin tissue, buoyant tissue,or unusual color can indicate that a tissue sample may yield fewerchondrocytes after tissue processing.

In some embodiments, a source cell preparation is prepared from a tissuesample within about 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days,5 days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks following collectionof a tissue sample from a human subject. In some embodiments, a tissuesample is subject to processing. In some embodiments, chondrocytes areisolated from a tissue sample. In some embodiments, a tissue sample istransported in transport media. In some embodiments, a tissue sample iscapable of being stored in transport media for up to seven daysfollowing procurement. In some embodiments, transport media is decantedaway from a tissue sample. In some embodiments, transport media isaspirated away from a tissue sample.

In some embodiments, once a tissue sample is isolated, any bone andsynovium, if present, are trimmed away. In some embodiments, aftertrimming, the target final weight of a tissue sample is less than orequal to 9 g. In some embodiments, trimmed a tissue sample weighingbetween about 1 g and about 9 g is processed. In some embodiments,trimmed a tissue sample weighing about 9 g is processed. In someembodiments, trimmed a tissue sample weighing less than 1 g is notprocessed. In some embodiments, trimmed a tissue sample weighing morethan 9 g is not processed. In some embodiments, trimmed a tissue sampleis divided into approximately 300 mg samples. In some embodiments, atissue sample is divided into samples weighing between about 200 mg andabout 400 mg. In some embodiments a tissue sample is divided intosamples weighing between about 250 mg and about 320 mg. In someembodiments, a tissue sample is divided into samples weighing betweenabout 280 mg and about 300 mg. In some embodiments, a tissue sampleweighs about 300 mg. In some embodiments a tissue sample does not weighless than about 280 mg. In some embodiments, each tissue sample isprocessed separately.

In some embodiments, a tissue sample is subjected to mechanicaldisruption to release cells. In some embodiments, prior to enzymatictreatment, tissue is minced to aid in digestion. In some embodiments,tissue is minced into pieces of about 2 mm² to about 3 mm². In someembodiments, tissue is minced into pieces of about 1 mm² to about 2 mm².In some embodiments, tissue is minced into pieces of about 0.5 mm² toabout 1 mm². In some embodiments, tissue is minced into pieces of about0.5 mm².

In some embodiments a tissue sample is subjected to enzymatic treatment.In some embodiments, a tissue sample is be washed and incubated in acell growth medium containing an enzyme(s) to digest tissue surroundingthe cells without damaging the cells. In some embodiments, tissue isdigested using a combination of a non-specific protease and collagenase.In some embodiments, tissue comprising chondrocytes may be digestedusing cell growth medium comprising a non-specific protease. In someembodiments, tissue is digested for about 30 minutes to about 90minutes, about 30 minutes to about 2 hours, about 60 minutes to about 90minutes or about 60 minutes to about 2 hours at 37° C. In someembodiments, a cartilage sample is digested in non-specific protease for60-90 minutes. Following treatment with a non-specific protease, in someembodiments, tissue is further digested with collagenase in cell growthmedium. In some embodiments, tissue is digested for about 8 hours toabout 16 hours, about 8 hours to about 24 hours, about 8 hours to about32 hours, about 16 hours to about 24 hours or about 16 hours to about 32hours at 37° C. In some embodiments, a cartilage sample is digested incollagenase for about 16 to 24 hours. In some embodiments, tissue isdigested in a 5% CO₂ atmosphere. In some embodiments, tissue is digestedin a closed container. In some embodiments, cell growth medium includesDulbecco's Modified Eagle Medium (DMEM), about 20% (+/−1%) irradiatedfetal bovine serum (irFBS), and optionally an antibiotic (e.g., 40 μg/mLgentamicin), an antifungal, and factor(s) for induction of lineage celldifferentiation (hereinafter “cell growth medium”). In some embodiments,cell growth medium includes ascorbic acid and or transforming growthfactor-β (TGF-β). In some embodiments, cell growth medium includesHEPES. In some embodiments, cell growth medium includes glutamax. Insome embodiments, cell growth medium includes high glucose. In someembodiments, cell growth medium includes L-glutamine. In someembodiments, cell growth medium comprises DMEM, HEPES, Fetal BovineSerum, and Gentamicin

In some embodiments, following steps of digestion, cells are isolated bycentrifugation. In some embodiments, supernatant is decanted away fromthe cell pellet. In some embodiments, supernatant is aspirated away fromthe cell pellet. In some embodiments, centrifugation is followed bywashing with cells with growth medium. In some embodiments, the cellpellet is re-suspended in growth medium.

In some embodiments, isolated cells are counted and assessed forviability. In some embodiments, cells are counted with a hemacytometer.In some embodiments, viable cells are counted. In some embodiments,non-viable cells are counted. In some embodiments viable cells per mLare calculated.

In some embodiments, following isolation, cells are cultured in cellgrowth medium for about 3 days to about six weeks, at 37° C. in a 5% CO₂atmosphere. The culture period may vary depending upon the number ofcells initially obtained. Culturing time may vary with different celltypes since different cell types have different rates of proliferation.

In some embodiments, medium that supports proliferation and/ordifferentiation of cells in tissue culture is utilized. One of ordinaryskill in that art will be aware of a variety of potentially useful mediaincluding for example, Dulbecco's Modified Eagle's Medium (DMEM),α-modified Minimal Essential Medium (α-MEM), and Roswell Park MemorialInstitute Media 1640 (RPMI Media 1640) and the like. In someembodiments, up to about 20% Fetal Bovine Serum (FBS) or 1% to 20% horseserum is added to medium to support proliferation of chondrocytes.

In some embodiments, a defined medium may be used; in some suchembodiments, one or more growth factors, cytokines, hormones and FBS isprovided at appropriate concentrations to permit and/or facilitate cellgrowth, proliferation, and/or differentiation.

In some embodiments, cells are grown and/or maintained at temperaturesbetween 31° C. and 37° C. with a CO₂ content between 2% and 10% and anoxygen content between 1% and 22%. In some embodiments, cells may bemaintained under these conditions for up to 6 weeks.

In some embodiments, cells are maintained in culture for up to 2passages, 3 passages, 4 passages, 5 passages, 6 passages, 7 passages, 8passages, 9 passages, 10 passages or more.

In some embodiments, chondrocytes de-differentiate in monolayer cellculture, exhibiting a fibroblastic phenotype and down regulation of Col2and Aggrecan. Without wishing to be bound by theory, de-differentiationoccurs after removal of chondrocytes from a 3-dimensional cartilagematrix and is observed during proliferation and expansion of cells inmonolayer culture.

In some embodiments, cells are cultured to a density sufficient toachieve a cell seeding density as described herein.

In some embodiments, cells are contacted with an enzyme (e.g., trypsin)for a period of time sufficient to release cells from a tissue culturesurface. In some embodiments, released cells are provided as asuspension of cells. In some embodiments, a suspension of cells isbrought into contact with one or more predetermined portions of asupport matrix, (e.g., with one surface, a portion of a surface), suchthat a substantial portion of cells, or substantially all cells, contactone or more surfaces of a support matrix. In some embodiments, cells areretained only on one surface or an edge of one surface of a supportmatrix. In some embodiments, upon contact with a support matrix cellsadhere.

In some embodiments, a support matrix (e.g., resorbable collagenmembrane) is provided in a container. In some embodiments, a supportmatrix is placed or positioned over a bottom surface of a container. Insome embodiments, a container comprises an anchor. In some embodiments,a support matrix is placed or positioned over a bottom surface of acontainer and an anchor is place on top of such support matrix. One ofordinary skill will be aware that an anchor placed on top of a supportmatrix may contact only an outer edge (e.g., peripheral edge) of asupport matrix. In some embodiments, an anchor placed on top of asupport matrix defines an area or volume bounded by sidewalls of suchanchor and top surface of a support matrix. In some embodiments, an areaor volume bounded by sidewalls of an anchor and top surface of a supportmatrix confines cells (e.g., chondrocytes) within such area or volume.In some embodiments, a cell seeded support matrix is secured within acontainer comprising an anchor.

In some embodiments, such controlled seeding of cells on and/or near asupport matrix surface may allow cells to freely migrate and/or populatea site of implant, and/or may lead to enhanced cell proliferation and/orregeneration of tissue at a site of implant.

As presently disclosed, uniform seeding is preferable. Without wishingto be bound by theory, it is believed that the number of cells seededdoes not limit an amount of final tissue produced in a patient treatedusing a cell seeded support matrix. However optimal seeding may increasea rate of cell and or tissue generation in a patient following implant.In some embodiments, optimal seeding amounts depend on cultureconditions. In some embodiments, uniform seeding is characterized by alack of gaps or space between seeded cells.

In some embodiments, chondrocytes are seeded on a support matrix (e.g.,porcine type I and III collagen) at a density of at least about 250,000cells/cm², 300,000 cells/cm², 400,000 cells/cm², 500,000 cells/cm²,600,000 cells/cm², 700,000 cells/cm², 800,000 cells/cm², 900,000cells/cm², 1,000,000 cells/cm², or more. In some embodiments, cellsseeded on a support matrix may divide and/or differentiate.

In some embodiments, a total of at least 5×10⁶, 7.5×10⁶, 1.0×10⁷,1.5×10⁷, 2.0×10⁷, 2.5×10⁷, 3.0×10⁷ or more chondrocytes are seeded onsupport matrix (e.g., type I and III collagen). In some embodiments, atotal of about 1.0×10⁷ chondrocytes to about 2.0×10⁷ chondrocytes areseeded on a 20 cm² porcine type I and type III collagen membrane. Insome embodiments, a total of about 7.5×10⁶ chondrocytes to about 1.5×10⁷chondrocytes are seeded on a 14.5 cm² porcine type I and type IIIcollagen membrane.

In some embodiments, a cell seeded support matrix is contacted withmedium favorable to cell growth, adherence to a support matrix,viability and/or differentiation. In some embodiments, a mediumcomprises DMEM. In some embodiments, a medium is supplemented withsubstances, e.g., fetal bovine serum, antibiotic (e.g., gentamicin). Insome embodiments, medium is supplemented with about 7%, about 8%, about9%, about 10%, about 11% fetal bovine serum. In some embodiments, mediumis supplemented with 8.9%+/−0.2% fetal bovine serum. In someembodiments, a membrane comprising porcine collagen type I and IIIseeded with allogeneic chondrocytes is contacted with DMEM supplementedwith 8.9%+/−0.2% FBS and 45 μg/mL gentamicin. In some embodiments, cellsseeded on a support matrix are contacted with medium as described abovefor at least 1, 2, 3, 4, 5, 6, 7, 8 or more days. In some embodiments,chondrocytes seeded on a support matrix are contacted with medium asdescribed above for about 2 to 4 days.

In some embodiments, a quality assessment step is performed on a cellseeded support matrix, or a portion thereof. In some embodiments, aquality assessment step comprises evaluation or measurement of membraneintegrity, presence or absence of particulate matter, cell viability,cell density, cell identity and or sterility.

In some embodiments, medium containing supplements is removed fromchondrocytes seeded on a support matrix by a step comprising rinsing. Insome embodiments, a rinsing step is a series of rising steps. In someembodiments, a cell seeded matrix is rinsed using medium, for example,phenol red free DMEM. In some embodiments, phenol red free DMEM is alsosuitable as a storage or shipping medium.

In some embodiments, a biocompatible adhesive or glue is utilized forcontacting cells with a support matrix. In some embodiments, cells aremixed with a biocompatible adhesive or glue before, during and or aftercontact with a support matrix. In some embodiments, a biocompatibleadhesive or glue may be layered over cells on a support matrix, belowcells on a support matrix or impregnated within a support matrix.

In some embodiments, the present disclosure provides for combining cellsand glue combined together in a mixture and forming one or morealternating layers of cells and glue on a surface or edge of a supportmatrix.

In some embodiments, biocompatible adhesives or glues used incompositions of the disclosure include an organic fibrin glue (e.g.,TISSEEL®, fibrin based adhesive available from Baxter, Austria) or afibrin glue prepared during surgery using autologous blood.

Methods and Uses of Cell Bank Preparation

The present disclosure contemplates storage and banking of culturedcells and/or cell seeded support matrices for later use. Alsocontemplated are methods of use of stored cells.

In some embodiments, a source tissue sample obtained from a single humansubject provides sufficient numbers of chondrocytes, and/or chondrocyteprecursors, to treat multiple other human subjects. In some embodiments,enough source tissue may be obtained from a single subject to providesufficient chondrocytes to treat about 100, about 200, about 500, about1000, about 1500 or about 2000 subjects.

Typically, a source tissue sample is obtained from a tissue comprisingchondrocytes or chondrocyte precursors. For example, chondrocytes andchondrocyte precursors may be isolated from cartilage (e.g., hyalinecartilage, fibrocartilage or elastic cartilage) or bone marrow. In someembodiments, suitable cartilage may be located at an articular surface(e.g., knee joint) or at a meniscus. In some embodiments, suitablecartilage may be located at a femoral condyle, in particular at asuperior-external region, or a lateral external region of an incisura.In some embodiments, chondrocytes isolated from a source tissue sampleobtained from an articular surface are suitable for preparation ofcompositions described herein.

In some embodiments, a source tissue (e.g., a source cartilage) isobtained from a site in a source subject that corresponds to a lesionsite in a recipient subject.

In some embodiments, cultured cells and/or cell seeded support matricesare stored at about 0° C., about 4° C., about 6° C., about 8° C., about10° C., about 12° C., about 14° C., about 16° C., about 18° C., about20° C., about 22° C., about 24° C., about 26° C., about 28° C., about30° C., about 32° C., about 34° C., about 36° C., about 37° C. orhigher. In some embodiments, cultured cells and/or cell seeded supportmatrices are stored at less than 0° C. In some embodiments, culturedcells and/or cell seeded support matrices are stored at about −5° C. toabout 5° C., about 5° C. to about 10° C., about 10° C. to about 15° C.,about 15° C. to about 20° C., about 20° C. to about 25° C., about 25° C.to about 30° C., about 30° C. to about 35° C., about 35° C. to about 40°C. In some embodiments, cultured cells and/or cell seeded supportmatrices are store at about −5° C. to about 37° C., and ranges therein.Without wishing to be bound by theory, storage advantageously enableschondrocytes to be conserved for long periods, without affecting theirfunctional properties. In some embodiments, chondrocyte preparationsand/or chondrocyte seeded collagen membranes may be stored underconditions described herein, for example, prior to in vivo implantation.

In some embodiments, a cell preparation and/or cell seeded supportmatrix described herein is stored for at least 24 hours, at least 2days, at least 5 days, at least 7 days, at least 14 days, at least 21days, at least 28 days, at least 1 month, at least 6 months, at least 12months or longer.

In some embodiments, cells are stored as a primary cell bank (e.g.,following an initial culture step). In some embodiments, cells in aprimary cell are stored for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10years. In some embodiments, cells are stored as a secondary, or workingcell bank. A secondary, or working cell bank may be comprised of cellsderived from a primary cell bank.

In some embodiments, cells are treated with trypsin prior tocryopreservation. In some embodiments, cells are treated with trypsin ata confluence level between about 50% to about 100%. In some embodiments,cells are treated with trypsin at a confluence level between about 50%to about 90%. In some embodiments, cells are treated with trypsin at aconfluence level at about 70%. Alternatively, in some embodiments, cellsare treated with trypsin when primary cultures have reached a maximum of16 days of growth and when secondary cultures have reached a maximum of10 days of growth. In some embodiments, cells are treated with 0.05%trypsin and EDTA. In some embodiments, after treatment with trypsin,cells are analyzed for viability using a hemacytometer.

In some embodiments, cells are frozen at a cell density between about1×10⁶ cells/mL. and about 5×10⁶ cells/mL. In some embodiments, 20% DMSOis added to a cryopreservation vial at a volume equal to the volume ofcells in the vial. In some embodiments, 20% DMSO is added to thecryopreservation vial drop-wise. In some embodiments, cells are frozenat a temperature between about −70° C. and about −60° C. In someembodiments, cells are frozen at a temperature at about −80° C. In someembodiments, cells are stored long term in liquid nitrogen vapor (LN₂).In some embodiments, cryopreservation vials are transferred to LN₂ afterbeing frozen at about −80° C. for between about 2 hours and about 24hours. In some embodiments, cells are stored long term for between about1 day and about 7 years.

In some embodiments, a saline solution (e.g., a solution isotonic withplasma) is used to store chondrocytes. In some embodiments, cells orcell seeded support matrix may be stored in a solution comprisingchloride salts (e.g., sodium chloride, potassium chloride, calciumchloride and/or magnesium chloride) and lactates (e.g., sodium lactate).In some embodiments, an isotonic saline solution may comprise sodiumchloride, potassium chloride, magnesium chloride and sodium lactate. Insome embodiments, an isotonic saline solution may comprise sodiumchloride, potassium chloride, calcium chloride and sodium lactate, whichis equivalent to a “Ringer-lactate solution.”

In some embodiments, cells to be stored are analyzed for qualitycontrol. In some embodiments, thawed cell bank cells are analyzed forquality control. In some embodiments, primary cells are analyzed forquality control. In some embodiments, secondary cells are analyzed forquality control. In some embodiments, tertiary cells are analyzed forquality control. In some embodiments, cells are analyzed for mycoplasma.In some embodiments, cells are analyzed for endotoxins. In someembodiments, cells are analyzed for sterility. In some embodiments,cells are analyzed for viruses. In some embodiments, cells are analyzedfor retroviruses. In some embodiments, cells are analyzed for karyology.In some embodiments, cells are analyzed for cell line identification(e.g. chondrocytes). In some embodiments, cells are analyzed forviability. In some embodiments, cells are analyzed for senescence. Insome embodiments, cells are analyzed for confluence.

In some embodiments, cryogenically frozen cells are thawed. In someembodiments, cryogenically frozen cells are thawed for cultureinoculation. In some embodiments, cells are thawed at between about35.5° C. and about 38.5° C. In some embodiments, cells are thawed atabout 37° C. In some embodiments, cells are thawed in a heat block. Insome embodiments, cells are thawed in a digital dry bath unit. In someembodiments, cells are thawed in a water bath. In some embodimentsthawed cells are moved to a new tube. In some embodiments thecryopreservation vial is washed with media. In some embodiments the washis added drop-wise to the thawed cells.

In some embodiments, thawed cells are measured for viability. In someembodiments, viability is measured with a hemacytometer. In someembodiments, at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or more of cellspresent are viable. In some embodiments, at least 90% of cells in apreparation are viable. In some embodiments at least 70% of cells in apreparation are viable.

In some embodiments percent recovery after thaw is calculated. In someembodiments the percent recovery is at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98% or more. In some embodiments, at least 50%of cells are recovered after thaw.

Methods of Treatment

In some embodiments, the present disclosure contemplates use ofchondrocytes seeded and grown on a support matrix (e.g., collagenmembrane) to treat/repair cartilage defects, lesions and/or injuries ina subject. Alternatively or additionally, in some embodiments, thepresent disclosure contemplates use of compositions disclosed herein toregenerate cartilage in a subject. In some embodiments, cartilagedefects, lesions and/or injuries are located in an articulating joint(for example, knee, ankle, elbow, shoulder, hip, or wrist) of a subject.In some embodiments, a defect in a medial femoral condyle, a lateralfemoral condyle or trochlea of a subject is/are treated usingcompositions of the present disclosure.

In some embodiments, a subject who is treated is an adult human. In someembodiments, a subject who is treated is a human between 10 to 17 yearsin age; in some such embodiments, a subject does not have an open growthplate.

In some embodiments, when a cell seeded support matrix is implanted at asite of a defect, lesion and/or injury, a matrix is placed with cellsfacing (e.g., in contact with) a surface to be treated. In someembodiments, a cell seeded support matrix is implanted into, and/orover, a site of a lesion, defect and/or injury. In some embodiments, acell seeded support matrix is provided in a form (e.g., a sheet form)that is readily shaped (e.g., by folding, cutting, trimming etc.) foradministration to a chondral or osteochondral defect. In someembodiments, a cell seeded support matrix is shaped into a form thatuniquely fits or adheres to a subject's chondral or osteochondraldefect.

In some embodiments, after a cell seeded support matrix is implantedinto a defect, lesion and/or injury, a covering matrix is secured usinge.g., a biocompatible adhesive or suture. In some embodiments, acovering matrix serves to cover an area to prevent infiltration ofundesirable cells and/or biological factors (e.g., fibroblasts,macrophages) from surrounding tissue into an area to be treated. In someembodiments, a covering matrix comprises any support matrices describedherein, and/or may include hyaluronic acid, fibrin and/or polylacticacid. In some embodiments, a covering matrix is cell-free andresorbable. In some embodiments, a covering matrix is semi-permeable.

In some embodiments, biocompatible adhesives or glues used to secure acovering matrix include an organic fibrin glue or sealant (e.g.,TISSEEL®, fibrin based adhesive available from Baxter, Austria) or afibrin glue prepared during surgery using autologous blood.

In some embodiments, a biocompatible adhesive or glue is applied to adefect prior to placement of a cell seeded support matrix over, or into,a defect. In some embodiments, a biocompatible adhesive or glue isapplied to a cell seeded support matrix prior to placement over, orinto, a defect. In some embodiments, a biocompatible adhesive or glue isapplied to a periphery of an implant.

In some embodiments, a cell seeded support matrix is injected into asite of implantation, with or without an adhesive or glue. In someembodiments, a cell seeded support matrix is implanted via a minimallyinvasive procedure. In some embodiments, a minimally invasive procedureconsists of arthrotomy, mini-arthrotomy, arthroplasty and arthroscopy.

In some embodiments, one or more cell seeded support matrices areimplanted to treat a region comprising a defect, lesion and/or injury.In some embodiments, 1, 2, 3, 4, 5 or more cell seeded support matricesare implanted in a region comprising a defect, lesion and/or injury. Insome embodiments, more than one cell seeded support matrix is layeredinto, or over, a defect, lesion and/or injury.

In some embodiments, a single matrix is utilized to treat multipledefects. In some embodiments, a plurality of defects is treated, eachwith a different matrix. In some embodiments, one or more defects istreated with a plurality of individual matrices.

In some embodiments, following treatment with a composition of thepresent disclosure, a region treated (e.g., an articular joint) isevaluated using a screening method (e.g., magnetic resonance imaging).In some embodiments, a treated region is evaluated for filling, repairand/or healing of a defect, lesion and/or injury.

All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference in their entirety. Inaddition, the materials, methods and examples are illustrative only andnot intended to be limiting. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this disclosurebelongs. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentdisclosure, suitable methods and materials are described herein.

The disclosure is further illustrated by the following examples. Theexamples are provided for illustrative purposes only. They are not to beconstrued as limiting the scope or content of the disclosure in any way.

EXAMPLES Example 1: Knee Cartilage Repair Pre-Clinical Feasibility StudyComparing Autologous and Allogeneic Chondrocyte Sources Combined withMaci Procedure

All procedures were conducted after IACUC approval following ASTMF2451-05(2010)1. A total of 18 skeletally mature New Zealand whiterabbits were used to compare autologous versus allogeneic chondrocytesources. An additional 8 rabbits were used for controls (defect only andcollagen membrane only, 4 rabbits per group).

For the autologous versus allogeneic study, rabbits were divided intothe following cohorts: 1-6: autologous group; 7-12: donors forallogeneic group (non-survival, cartilage harvest only); 13-18:allogeneic group.

Knee cartilage was harvested from rabbits. Collected cartilage tissuewas placed in 10 ml Falcon tubes in 0.9% NaCl and transported to VericelCorporation (Cambridge, MA) for cartilage tissue digestion and cellisolation. Cell-seeded collagen membranes were ready for implantationapproximately 3 weeks later. Each sheet contained approximately500,000-1,000,000 cells per cm².

NZWR 1-6 received autologous MACI implants on the knee (trochleargroove), while NZWR 13-18 received MACI implants using allogeneicchondrocyte sources. For each rabbit, two 3 mm diameter chondral defectswere created in the trochlear groove: one in the superior aspect of thegroove, and a second defect in the inferior aspect of the groove,superior to the intercondylar notch. The MACI membrane was cut out usinga 3 mm biopsy dermal punch, placed over the defects, and secured usingfibrin gel sealant (Tisseel®, Baxter). The patella was carefullyrelocated into the trochlear groove, the knee capsule was closed using3-0 Nylon, and dermal tissues were closed using 3-0 Vicryl. Aftersurgery, the rabbits were returned to their cages and kept in anenvironment with controlled temperature, humidity and circadian cycle,receiving food and water ad libitum. All rabbits were euthanized at 12weeks. Knees were harvested and processed for histological analysis.Hematoxylin and eosin (H&E), toluidine blue, and collagen type IIstaining was performed on deparaffinized slides to evaluate neocartilageformation.

All NZWRs reached the 12-week time-point. Results are summarized in FIG.1 . No cartilage regeneration was grossly or histologically visualizedin control (defect only group) and membrane-only groups. Only theresiduals of collagen membranes were still present in the collagenmembrane only group. In the autologous cohort, gross examinationrevealed defects covered by hyaline-like cartilage tissue. Synovialfluid was clear and present in normal amounts. Histologically, cartilagetissue was characterized by abundant toluidine blue staining. Collagentype II staining matched the glycosaminoglycan (GAG) staining patterns.

At 12 weeks, the neocartilage tissue partially integrated with thesurrounding native cartilage tissue. There were no signs of inflammatoryinfiltrate within the osteochondral tissues. In the allogeneic group,grossly, defects were covered by hyaline-like cartilage tissue. Therewere no signs of infection or inflammation, and synovial fluid was clearpresent in normal amounts. Histologically, defects were filled withcartilage tissue that displayed abundant GAG deposition via toluidineblue staining. Collagen staining followed the same staining pattern.Similarly to the autologous group, the neocartilage was partiallyintegrated with the surrounding cartilage. Importantly, no evidentimmune response including angiogenesis or infiltrating of immunologicalcells showed in the adjacent tissues.

Example 2: Preparation of Cell Bank Samples

This example demonstrates culturing and freezing cells for long-termstorage in a cell bank for use in future subjects.

Before processing, all tissue samples are visually inspected for clarityand accuracy.

If tissue is received in transport media, the media is aspirated awayand discarded. If tissue is received in a bag, sterile scissors are usedto cut the top corner of the bag. Transport media is aspirated, and thenthe bag is cut open and pulled apart to create a flat, sterile field.Tissue is transferred to a sterile petri dish and rinsed with Ham's F-12mixture with L-glutamine (e.g. F-12 solution).

The tissue is weighed and visually inspected for synovium, bone, or anyother unwanted tissue by prodding each side to detect any areas thatlack rigidity, appear fibrous, or appear fatty. Tissue is trimmed of allbone and synovium so that only cartilage remains. The tissue is thenweighed again. If trimmed weight is greater than 9 g, remove excesstissue until the final weight is less than or equal to 9 g. Tissuegreater than 9 g or less than 1 g should not be processed.

The weight of the tissue sample is divided by 300 mg to determine howmany centrifuge tubes are needed for processing. In each tube, 1×non-specific protease solution is added by adding 5 mL of F-12 solutionand 5 mL of 2× protease to each tube. The tissue is divided into piecesweighing between 280 mg and 300 mg and each piece is placed into asterile petri dish. 0.5 mL of F-12 solution is added to each piece oftissue to ensure the pieces do not dry out. The F-12 solution isaspirated away when ready for protease digestion.

0.5 mL of 1× protease/F-12 solution from each centrifuge tube is addedto each petri dish. The tissue is finely minced until all of the piecesare smaller than approximately 0.5 mm². The minced tissue is transferredto centrifuge tubes comprising 1× protease/F-12 solution. The reactionsare incubated at 37° C.±1.5° C. for 75 minutes±15 minutes.

After incubation, the 1× protease/F-12 solution is removed. The tissuepieces are rinsed with 10 mL of F-12 solution. 9 mL of DMEM withL-glutamine, high glucose, and HEPES (e.g. EXXXX solution) and 1 mL of1% collagenase is added to each centrifuge tube and the reactions areincubated at 37° C.±1.5° C. for 20 hours±4 hours.

10 mL of DMEM with HEPES, glutamax, 20% irradiated Fetal Bovine Serum,and 40 μL/mL gentamicin (e.g. EG2MX solution) is added to each tube.Suspensions from each tube are pooled and centrifuged at 210 RCF for 5minutes. The supernatant is aspirated away and the pellet isre-suspended in 5 mL EG2MX solution.

Cells are counted for viability using a hemacytometer. Using amicroscope, the middle square and each corner square are counted. Viableand non-viable cells are counted and the number of viable cells/mL iscalculated. The number of cells needed to prepare to seed at a densityof 5×10⁵ cells/flask in 35 mL of EG2MX solution is determined. Cells aregrown in a 5% CO₂ incubator at 37° C.±1.5° C.

Primary cells are fed between 3 and 5 days after initiation of cultureand in 1 to 4 days intervals afterwards. Conditioned media is pouredfrom each flask into a waste container and 40 mL EG2MX is added intoeach flask.

Secondary cells are fed between 1 and 4 days after initiation of cultureand in 1 to 4 day intervals afterwards.

Prior to cryopreservation, conditioned media is analyzed for qualitycontrol. Samples are taken to analyze for sterility.

Example 3: Cryopreservation of Cell Cultures for Storage in Cell Bank

This example demonstrates cryopreservation of allogeneic cell culturefor preservation in a cell bank.

Cells are treated with trypsin prior to storage once cells are between70%±20% confluence or when cultures have reached a maximum of 16 days ofgrowth for primary cultures or 10 days for secondary cultures.

Conditioned media is removed from the flask and then each flask isrinsed with EDTA.

0.05% trypsin and EDTA solution is added to the flask and incubated at37° C.±1.5° C. until cells are detached, and for no longer than 8minutes. 10 mL of EG2MX solution is added to deactivate trypsin.

The suspension is centrifuged at 210 RCF for 5 minutes. The supernatantis poured off and the pellet is re-suspended with 3 mL EG2MX.

Cells are counted for viability using a hemacytometer. Using amicroscope, the middle square and each corner square are counted. Viableand non-viable cells are counted and the number of viable cells/mL iscalculated. The volume needed to prepare a suspension of 10×10⁶ cells isdetermined, add that volume of EG2MX is added to the tube. Thesuspension is now at 5×10⁶ cells.

The same volume of 20% DMSO as there is volume of suspension is added toeach tube. Then, 1 mL of this suspension is added to a cryopreservationvial. The suspension is frozen at −80° C.±10° C. for at least 2 hours,and up to 24 hours.

After initial freeze, cryopreservation vials are transferred to acryogenic storage dewar comprising liquid nitrogen (LN₂).

Example 4: Use of Cell Bank

This example demonstrates thawing of cell bank samples for use incharacterization assays and for products.

The frozen cryopreservation vials are placed in a heat block set at 37°C.±1.5° C. for approximately 5 minutes.

Once thawed, the contents are transferred to a new tube. Thecryopreservation vial is rinsed with EG2MX solution and transferred tothe new tube. The volume is increased to 10 mL with EG2MX.

Cells are counted for viability using a hemacytometer. Using amicroscope, the middle square and each corner square are counted. Viableand non-viable cells are counted and the number of viable cells/mL iscalculated. The percent of cells recovered after thaw is calculated. Thepercent recovery should be at least 50%.

To initiate cell culture, 35 mL of EG2MX solution is inoculated with 5mL of cell suspension in a flask. Cells are grown in a 5% CO₂ incubatorat 37° C.±1.5° C.

Example 5: Characterization of Cell Bank

This example demonstrates assays used to characterize and/or determinequality of primary (master) cell bank samples prior to use for aproduct.

After thawing a primary or master cryopreservation vial of cells, cellsare counted for viability using a hemacytometer. Using a microscope, themiddle square and each corner square are counted. Viable and non-viablecells are counted and the number of viable cells/mL is calculated. Thevolume of cell suspension needed to achieve 1×10⁵ cells/mL iscalculated. This volume is used for agarose, pellet culture, six wellplate, identity/aggrecan, and senescence assays from secondary cultures.

Flasks of 5×10⁵ and 1×10⁵ primary cells are prepared for secondary cellculture and grown in a 5% CO₂ incubator at 37° C.±1.5° C. Feedings aredone between 1 to 4 days after culture initiation. Cells are treatedwith trypsin as described in Example 3. Trypsin treated cells are usedfor agarose, pellet culture, and six well plate quality control assays.

Secondary cells are once again counted for viability and flasks withEG2MX are inoculated with cell suspension to obtain 8.0×10⁵ cells/flaskto create a tertiary cell culture. Cells are grown in a 5% CO₂ incubatorat 37° C.±1.5° C. Tertiary cultures are used to characterize cell banksamples for identity (e.g. presence of chondrocytes) and the presence ofaggrecan. The volume of secondary culture suspension needed to achieve1×10⁶ cells/tube for agarose testing and six well plate testing iscalculated. Tertiary cultures are fed between 1 and 4 days afterinitiation of culture and in 1 to 4 day intervals afterwards.

For identity and aggrecan testing, cells are loaded onto a membrane.Prior to loading onto a membrane, tertiary cultures are treated withtrypsin as described in Example 3. Flasks are pooled and the suspensionsare rinsed through a strainer. Cells are counted for viability using ahemacytometer. 20.00×10⁶ cells in 25 mL are required for membraneloading. The cells are centrifuged at 210 RCF for 5 minutes and thesupernatant is removed.

The membrane is placed rough side up in a petri dish and wet with DMEMcontaining HEPES, high glucose, glutamax, 45 μg/mL gentamicin, and8.9%±0.2% FBS (e.g. EG1MX solution). The cell pellet is re-suspended in5 mL of EG1MX, and the volume is brought to a final volume of 25 mL. Themembrane is placed in a 5% CO₂ incubator at 37° C.±1.5° C. The membraneis packaged 2 to 4 days after loading and rinsed with 15 mL of DMEM withHEPES, without phenol red (e.g. EXXIM).

Flasks of 1×10⁵ primary cells are prepared for senescence testing andgrown in a 5% CO₂ incubator at 37° C.±1.5° C. The cells must be treatedwith trypsin every 7 days as described in Example 3. Feedings occurbetween every 1 to 4 days after initiation of the culture.

Cells are counted for viability using a hemacytometer. Using amicroscope, the middle square and each corner square are counted. Viableand non-viable cells are counted and the number of viable cells/mL iscalculated. The volume of cell suspension needed to achieve 1×10⁵cells/mL is calculated. Flasks comprising EG2MX solution are inoculatedwith 1 mL of cells, grown in a 5% CO₂ incubator at 37° C.±1.5° C., andpopulation doublings are calculated for each flask, for each passage.PDL=[LOG(cell yield)−LOG(cells inoculated)]/LOG(2). Senescence isreached when the number of population doublings is less than 1 for twoweeks in a row.

If the cell bank sample has detectable mycoplasma, is positive forHIV-1, HIV-2, Hepatitis B, Hepatitis C, any other viral contaminants, ordoes not have a normal human karyotype, the sample will be discarded.

TABLE 1 Acceptance Criteria for Characterization Tests Quality ControlTest Acceptance Criteria Tissue type Intermediate bulk cartilage ornormal cartilage Donor screening (CQA) Meets AATB standards andregulations HIV Type 1 (HIV 1) Negative or Non-reactive HIV Type 2 (HIV2) Negative or Non-reactive Hepatitis B (HBV) Negative or Non-reactiveHepatitis C (HCV) Negative or Non-reactive Treponema pallidum (syphilis)Negative or Non-reactive Sterility (primary culture) No growthdetected/observed Endotoxin (primary culture) Less than or equal to 3EU/mL Mycoplasma (primary culture) No detectable mycoplasma Sterility(cell bank vial) No growth detected/observed Cell viability at primarythaw Greater than or equal to 70% viable cells (cell bank vial) Six wellplate Average cells/well is greater than or equal to 0.88 × 10⁵ cellsAgarose assay Greater than or equal to 6.8% of cells forming coloniesfor greater than or equal to 2 divisions Pellet culture dga greater thanor equal to −4.73 Ct and and RTPCR assays dg2 greater than or equal to−4.54 Ct Senescence Cultures do not senesce less than 5 passages or arenot immortal Identity d5L greater than or equal to 2.00 Ct Potency dgaCt greater than or equal to −10.00 Ct Detection of 14 Viruses by RTPCR(Panel I) No detectable viruses In vitro assay for the presence ofporcine No porcine viruses detected viruses 28-day in vitro assay forthe presence of No viral contaminants detected viral contaminants Testfor the inapparent viruses No adenovirus contamination detectedTransmission electron microscopic Report results examination of cellcultures Quantitative product enhanced reverse Negative transcriptaseassay for the detection of retrovirus in biological samples In vitroassay for the presence of bovine No bovine viruses detected viruses CO1barcode assay for cell line detection Identified as human Spectralkaryology analysis of human cell Normal human karyotype lines

EXEMPLARY EMBODIMENTS

-   -   Embodiment 1. A membrane comprising cultured allogeneic human        chondrocytes at a density of at least 250,000 cells per cm²,        wherein the cultured chondrocytes are derived from cryogenically        frozen cell bank samples; and wherein the cultured chondrocytes        are characterized by a quality control assay.    -   Embodiment 2. The composition of embodiment 1, wherein the cell        bank comprises human cadaver chondrocytes.    -   Embodiment 3. The composition of embodiment 1, wherein the cell        bank comprises primary cultures.    -   Embodiment 4. The composition of embodiment 1, wherein the cell        bank comprises secondary cultures.    -   Embodiment 5. The composition of embodiment 1, wherein the        quality control assay comprises one or more of agarose, six well        plate, and cell pellet assays.    -   Embodiment 6. A method of preparing a cell bank, the method        comprising steps of: obtaining a tissue sample from a human;        inspecting the tissue sample for contamination; weighing the        tissue sample; mincing the tissue sample; digesting the tissue        sample; counting cells in the tissue sample to determine        viability; culturing the cells; and cryogenically freezing the        cells for storage.    -   Embodiment 7. The method of embodiment 6 wherein the step of        obtaining a tissue sample comprises removing cartilage from a        human cadaver.    -   Embodiment 8. The method of embodiment 6 wherein the step of        obtaining a tissue sample comprises removing cartilage from a        human cadaver from within about 24 hours to within about 7 days        after death.    -   Embodiment 9. The method of embodiment 6 wherein the step of        obtaining a tissue sample comprises removing cartilage from a        human cadaver within about 24 hours after death.    -   Embodiment 10. The method of embodiment 6, wherein the tissue        sample is inspected for synovium, bone, fibrous tissue, fatty        tissue, and contamination.    -   Embodiment 11. The method of embodiment 6, wherein the tissue        sample weighs from about 1 g to about 9 g.    -   Embodiment 12. The method of embodiment 6, wherein the tissue        sample weighs about 9 g.    -   Embodiment 13. The method of embodiment 6, wherein the tissue        sample is divided into pieces weighing between about 200 mg and        about 400 mg.    -   Embodiment 14. The method of embodiment 6, wherein the tissue        sample is divided into pieces weighing between about 200 mg and        about 300 mg.    -   Embodiment 15. The method of embodiment 6, wherein the tissue        sample is divided into pieces weighing between about 280 mg and        about 300 mg.    -   Embodiment 16. The method of embodiment 6, wherein the tissue        sample is divided into pieces weighing about 300 mg.    -   Embodiment 17. The method of embodiment 6, wherein the tissue is        digested with a protease.    -   Embodiment 18. The method of embodiment 6, wherein the tissue is        digested with collagenase.    -   Embodiment 19. The method of embodiment 6, wherein the tissue        sample is minced into pieces from about 0.5 mm² to about 3 mm².    -   Embodiment 20. The method of embodiment 6, wherein the tissue        sample is minced into pieces from about 0.5 mm² to about 2 mm².    -   Embodiment 21. The method of embodiment 6, wherein the tissue        sample is minced into pieces from about 0.5 mm² to about 1 mm².    -   Embodiment 22. The method of embodiment 6, wherein the tissue        sample is minced into pieces of about 0.5 mm².    -   Embodiment 23. The method of embodiment 6, wherein the cells are        counted with a hemacytometer.    -   Embodiment 24. The method of embodiment 6, wherein the tissue        sample comprises from about 50% to about 100% viable cells.    -   Embodiment 25. The method of embodiment 6, wherein the tissue        sample comprises from about 70% to about 100% viable cells.    -   Embodiment 26. The method of embodiment 6, wherein the tissue        sample comprises about 70% viable cells.    -   Embodiment 27. The method of embodiment 6, wherein the cells are        cultured in medium comprising DMEM.    -   Embodiment 28. The method of embodiment 6, wherein the cells are        cultured at about 37° C.    -   Embodiment 29. The method of embodiment 6, wherein the cells are        cryogenically frozen at about −80° C. for about 2 to 24 hours.    -   Embodiment 30. The method of embodiment 6, wherein the cells are        cryogenically frozen and stored in liquid nitrogen.    -   Embodiment 31. A method of characterizing a cell bank sample,        the method comprising steps of: thawing cryogenically frozen        primary cell bank samples; culturing secondary samples from the        primary samples; culturing tertiary samples from the secondary        samples; and assaying the samples to determine cell viability,        mycoplasma, endotoxin, sterility, senescence, identity, and        aggrecan values.    -   Embodiment 32. A method of characterizing a cell bank sample        according to embodiment 31, wherein the samples are assayed        using an agarose assay.    -   Embodiment 33. A method of characterizing a cell bank sample        according to embodiment 32, wherein the agarose assay tests for        percentage of cells forming colonies for greater than or equal        to two divisions.    -   Embodiment 34. A method of characterizing a cell bank sample        according to embodiment 31, wherein an acceptance criterion of        the agarose assay comprises at least 6.8% of cells form colonies        for at least two divisions.    -   Embodiment 35. A method of characterizing a cell bank sample        according to embodiment 31, wherein the samples are assayed        using a six well plate assay.    -   Embodiment 36. A method of characterizing a cell bank sample        according to embodiment 35, wherein the six well plate assay        tests for an average number of cells per well.    -   Embodiment 37. A method of characterizing a cell bank sample        according to embodiment 36, wherein an acceptance criterion of        the six well assay comprises an average of cells per well that        is greater than or equal to 0.88×10⁵.    -   Embodiment 38. A method of characterizing a cell bank sample        according to embodiment 31, wherein the samples are assayed        using a pellet culture assay.    -   Embodiment 39. A method of characterizing a cell bank sample of        embodiment 31, wherein the pellet culture assay tests for        ability of cells to generate cartilage following cellular        culture expansion.    -   Embodiment 40. A method of characterizing a cell bank sample        according to embodiment 39, wherein an acceptance criterion of        the pellet culture assay comprises dga greater than or equal to        −4.73 Ct.    -   Embodiment 41. A method of characterizing a cell bank sample of        embodiment 31, wherein an acceptance criterion for the assay for        mycoplasma comprises no detectable level.    -   Embodiment 42. A method of characterizing a cell bank sample of        embodiment 31, wherein an acceptance criterion for the assay for        sterility comprises no detection of growth.    -   Embodiment 43. A method of characterizing a cell bank sample of        embodiment 31, wherein an acceptance criterion for cell        viability comprises at least 70% of cells are viable.    -   Embodiment 44. A method of characterizing a cell bank sample of        embodiment 31, wherein an acceptance criterion for senescence        comprises cultures do not senesce in less than 5 passages or        cultures are not immortal.    -   Embodiment 45. A method of characterizing a cell bank sample of        embodiment 31, wherein an acceptance criterion for identity        comprises identification of the presence of chondrocytes.    -   Embodiment 46. A method of culturing a cell bank sample, the        method comprising steps of: thawing a cryogenically frozen cell        bank sample; adding culture medium to a flask; counting cells to        determine viability; growing the cells and the medium in a cell        culture; feeding the cell culture; and treating the cell culture        with trypsin.    -   Embodiment 47. The method of culturing a cell bank sample of        embodiment 46, wherein the cryogenically frozen cell bank sample        is thawed in a water bath, a heat block, or a dry cell bath at        about 37° C.    -   Embodiment 48. The method of embodiment 46, wherein the sample        is cultured in medium comprising DMEM.    -   Embodiment 49. The method of embodiment 46, wherein the cells        are counted with a hemacytometer.    -   Embodiment 50. The method of embodiment 46, wherein the cell        culture is fed at least every 1 to 4 days.    -   Embodiment 51. The method of embodiment 46, wherein the cell        culture is treated with 0.05% trypsin solution.    -   Embodiment 52. The method of embodiment 46, wherein the cell        culture is treated with trypsin solution until the cells are        detached from the flask.

EQUIVALENTS

It is to be understood that while the disclosure has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

We claim:
 1. A composition comprising: cultured allogeneic cellscomprising chondrocytes grown from a cryogenically frozen cell banksample; and a resorbable collagen membrane comprising a combination oftype I and type Ill porcine collagen derived from porcine peritoneum;wherein the cultured allogeneic cells are seeded on the resorbablecollagen membrane at a density of at least 250,000 cells per cm²,wherein the allogeneic cells are arranged in a monolayer on theresorbable collagen membrane, and wherein the allogeneic cells arededifferentiated.
 2. A method of manufacturing an allogeneic cartilagematrix, the method comprising steps of: thawing a cryogenically frozencell bank sample comprising chondrocytes; culturing the cells of thesample; characterizing the sample; preparing a resorbable collagenmembrane; seeding the resorbable collagen membrane with the culturedcells; and packaging the resorbable collagen membrane, whereincharacterizing the sample comprises characterizing the sample by one ormore control assays comprising: an agarose assay comprising anacceptance criterion comprising at least 6.8% of cells forming coloniesfor at least two divisions.
 3. The composition of claim 1, wherein theallogeneic cells are configured to be implanted to treat a chondraldefect and/or an osteochondral defect.
 4. The composition of claim 3,wherein the allogeneic cells comprise cultured allogeneic cells grownfrom a cryogenically frozen cell bank sample, and wherein the cryogeniccell bank sample is thawed in a water bath, a heat block, or a dry cellbath at about 37° C.
 5. The composition of claim 4, wherein the culturedallogeneic cells are cultured in medium comprising DMEM.
 6. Thecomposition of claim 4, wherein the cultured allogeneic cells arecharacterized for sterility, endotoxin, mycoplasma, senescence,identity, aggrecan, and/or karyology.
 7. The method of claim 2, whereinthe matrix comprises dedifferentiated cells, wherein the density ofcells seeded on the resorbable collagen membrane is at least 250,000cells per cm², and wherein the matrix is coated with an adhesive agent.8. The composition of claim 1, wherein the cultured allogeneic cellscomprise chondrocytes, wherein the chondrocytes comprise humanchondrocytes that are (1) expanded in culture, and (2) seeded onto theresorbable collagen membrane, and wherein the composition comprises oneor more chondrocyte markers and one or more non-chondrocyte markers, thenon-chondrocyte markers comprising at least one of a fibroblast markerand a synoviocyte marker.
 9. The composition of claim 8, wherein thesupport matrix comprises at least 70% of viable cells, and wherein thecomposition comprises less than about 10 μg/ml of fetal bovine serumalbumin.
 10. The composition of claim 8, wherein the chondrocytes in thecell bank comprise tissue harvested from an adult human or tissueharvested from a cadaver.
 11. The composition of claim 10, wherein thetissue comprises chondrocyte precursors.
 12. The composition of claim 1,wherein the composition comprises at least one surface seeded withallogeneic human chondrocytes.
 13. The method of claim 2, whereinculturing the cells of the sample comprises culturing the cells attemperatures between 31° C. and 37° C., with a CO₂ content between 2%and 10%, and an oxygen content between 1% and 22%.
 14. The method ofclaim 2, wherein the one or more control assays comprises analyzingcells for bacterial endotoxins comprising an acceptance criterion ofless than or equal to 3 EU/mL.
 15. The method of claim 2, wherein theone or more control assays comprises an acceptance criterion forsenescence comprising cultures that do not senesce in less than 5passages.
 16. The method of claim 2, wherein the one or more controlassays comprises a pellet culture assay comprising an acceptancecriterion comprising: dga greater than or equal to −4.73 Ct; and dg2greater than or equal to −4.54 Ct.
 17. The composition of claim 1,wherein the cultured allogeneic cells are seeded on the resorbablecollagen membrane at a density of at least 1.5×10⁷ cells per cm².
 18. Acomposition comprising: cultured allogeneic cells comprisingchondrocytes grown from a cryogenically frozen cell bank sample; and aresorbable collagen membrane comprising a combination of type I and typeIII porcine collagen derived from porcine peritoneum, the resorbablecollagen membrane being coated with a fibrin adhesive; wherein thecultured allogeneic cells are seeded on the resorbable collagen membraneat a density of at least 250,000 cells per cm², wherein the allogeneiccells are dedifferentiated, and wherein the composition comprises atleast one of a chondrocyte marker and a non-chondrocyte marker.
 19. Themethod of claim 18, wherein the dedifferentiated allogeneic cells:exhibit a fibroblastic phenotype; down regulate expression of a geneencoding ECM; and/or produce and/or secrete a lesser amount of at leastone of collagen and aggrecan.